CN103923175A - Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate - Google Patents
Fluorescent substrate for detecting activity of trypsin acting on Cry1A protoxin and application of fluorescent substrate Download PDFInfo
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- CN103923175A CN103923175A CN201410155233.9A CN201410155233A CN103923175A CN 103923175 A CN103923175 A CN 103923175A CN 201410155233 A CN201410155233 A CN 201410155233A CN 103923175 A CN103923175 A CN 103923175A
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Abstract
The invention discloses a fluorescent substrate for detecting the activity of trypsin acting on Cry1A protoxin and an application of the fluorescent substrate. The fluorescent substrate consists of fluorescence quenching group-Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu-fluorescence group. The invention also discloses the application of the fluorescent polypeptide substrate R28 in detecting the activity of insect midgut trypsin which directly acts on activated bacillus thuringiensis Cry1A protoxin. Compared with a conventional protease detection method, the fluorescent substrate disclosed by the invention has definite specificity and pertinence for detecting the activity of the insect midgut trypsin which directly acts on the activated bacillus thuringiensis Cry1A protoxin and detecting the activity of the trypsin which only acts on the corresponding fluorescent substrate R28, and intense fluorescence signals are generated in an enzymatic hydrolysis reaction, so that the sensitivity of the detection is greatly improved.
Description
Technical field
The present invention relates to biotechnology, relate to a kind of fluorogenic substrate and application thereof for detection of acting on the trypsinase vigor of Cry1A class parent toxin, be specifically related to a kind of fluorogenic substrate and application thereof for detection of acting on the trypsinase vigor of bacillus thuringiensis (Bacillus thuringiensis) Cry1A class parent toxin.
Technical background
Bacillus thuringiensis (Bacillus thuringiensis) is a kind of Gram-positive, shaft-like, the soil bacteria that can form gemma.In its process of growth, be accompanied by the formation of gemma, cell energy synthetic proteins crystal, because these crystallins have toxic action to insect, thereby the insecticidal crystal protein that is otherwise known as (insecticidal crystal proteins, ICPs), wherein delta-endotoxin is a most important class insecticidal proteins.Delta-endotoxin is divided into again Cry toxin and Cyt toxin according to the difference of constructional feature and mechanism of action.
main according to the difference of insecticidal spectrum in 1989 with Whiteley, Cry toxin is divided into several large classes of CryI, CryII, CryIII, CryIV and CryV-IX, these dissimilar toxin are except insecticidal spectrum difference, also variant at aspects such as amino acid whose homology, molecular size ranges.Along with the new gene of Bt is constantly found, many new gene order homologies but insecticidal spectrum is different, or the identical but sequence of insecticidal spectrum homology not, adopt original naming system easily to cause confusion.Therefore, Crickmore etc. have revised Cry unnamed gene and the categorizing system of coding Bt toxin, unify take consensus amino acid sequence and evolutionary relationship as basis, carry out 4 grades of names.Cry1 represents 1 family (amino acid consistence surpasses 45%), Cry1A represents 1A subfamily (amino acid consistence surpasses 78%), Cry1Aa represents 1Aa gene (amino acid consistence surpasses 95%), Cry1Aa1 represents the 1st allelotrope (the Crickmore et al.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Mocrobiology and Molecular Biology Reviews of 1Aa gene, 1998,62 (3): 807-813).
Cry toxin protein roughly has following four steps to the toxic action of insect: (1), under the alkaline environment of insect midgut, crystallin is dissolved, discharges parent toxin; (2) midgut proteinase is processed to form activation toxin to parent toxin; (3) receptors bind on the brush border membrane vesicle (brush border membrane vesicles, BBMV) of activation toxin and midgut epithelial cells; (4) on midgut epithelial cells film, form hole, make lysis, osmotic pressure is unbalance, causes insect death.Cry1A class parent toxin be molecule amount at the parasporal crystal albumen of 130-140KD, by three structural domains, formed.The hydrophobic alpha-helix that structural domain I is held by N forms, a hydrophobic alpha-helix in the middle of being positioned at by other 6 amphipathic-helicals around, hole formation is relevant to this structural domain; Structural domain II is comprised of β-pleated sheet structure, and three parallel β-pleated sheet structures form asymmetric β prism, and one of them β-pleated sheet structure oppositely wraps up structural domain I, and toxin is relevant with this structural domain to receptors bind and pest-resistant specificity; Structural domain III is comprised of two antiparallel β-pleated sheet structures that are positioned at C end, and the structural domain III of alterable height is relevant to the receptors bind in insect midgut, desinsection specificity and Stability Analysis of Structures.But the essence element of this structure must just be brought into play follow-up toxic action after insect midgut protease digestion is the more stable active polypeptide in 65kDa left and right, reactivation process comprises the shearing that almost whole C holds and holds 25-30 amino acid cut at N, the digestion of Cry1Ac parent toxin has confirmed to occur in 28 arginine places of N end, now there are some researches show that trypsinase and Quimotrase have mainly participated in this process (Johnston et al.Resistance to Bacillus thuringiensis by the Indian meal moth, Plodia interpunctella:comparison of midgut proteinases from susceptible and resistant larvae.Journal of Invertebrate Pathology, 1990, 55 (2): 235-244).
Therefore, the activation of insect midgut protease hydrolyzed is the committed step that a toxin performance toxic action and insect contratoxin produce resistance, if it changes, can cause the activation capacity of parent toxin to reduce and the enhancing of contratoxin Degradation.Oppert etc. (1997) find after having studied the resistance Indian meal moth Midgut protein enzymic activity with Bte HD-198 screening, the generation of resistance is due to its activity decreased, the activation of parent toxin to be declined, and further research shows to have lacked a kind of important midgut proteinase in resistant strain.Forcada etc. (1996) also find because midgut proteinase is to the decline of Cry1Ab parent toxin activation and the rising to Cry1Ab Degradation on resistance Heliothis virescens CP73-3.In resistance small cabbage moth and European corn borer, it is also an important resistance mechanism that protease activated effect reduces.
The method of traditional research Midgut protein enzyme activity is generally to utilize pattern substrate to detect, as active for detection of total protease in azo-casein (azocasein), N-benzoyl-D, L-arginine p-nitrophenyl amine hydrochlorate (N-benzoyl-L-arginine-p-nitroanilide, BApNA) for detection of tryptic activity, N-succinyl--Ala-Ala-proline(Pro)-phenylalanine-p-Nitroaniline (N-succinyl-alanine-alanine-proline-phenylalanine-p-nitro anilide, SAAPFpNA) for detection of the activity of Quimotrase.Proteolytic enzyme is enzyme important in insect gut, for digest food and degraded, takes in the toxic substance in body.Different types of insect is due to the difference of host types and feeding habits, and the composition of proteolytic enzyme is also different, and substrate type and the ability of different proteasome degradations are all variant.In traditional measuring method, the information obtaining is about proteolytic enzyme, trypsinase or Quimotrase the enzymolysis ability to pattern substrate, not pointed, can not directly illustrate the effect of midgut proteinase to Bt toxin.And proteinase activity adopts for above-mentioned three pattern substrates in measuring is all color reactions, sensitivity is also lower.
Cry1Aa, Cry1Ab and Cry1Ac have the albumen of obvious insecticidal activity to lepidopterous insects in Cry1A toxoid, also be several most important active ingredients in Bt preparation, its gene is the current external source insecticidal protein gene mainly proceeding in cotton, corn and paddy rice, therefore study toxic action process and the resistance mechanism of insect to this proteinoid of Cry1A toxoid albumen, the effective utilization that from now on other is had to the Bt gene of insecticidal activity is had to good demonstration effect.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of fluorogenic substrate for detection of acting on the trypsinase vigor of Cry1A class parent toxin is provided.
Another object of the present invention is to provide the application of this substrate.
Another object of the present invention is to provide a kind of detection effect in the method for the trypsinase vigor of Cry1A class parent toxin.
Object of the present invention can be achieved through the following technical solutions:
A fluorescent polypeptide substrate R28 for detection of insect midgut trypsinase vigor, consists of: fluorescent quenching group-Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu-fluorophor.
Wherein, the described preferred Dabcyl of fluorescent quenching group and the described preferred Edans of fluorophor.
Fluorescent polypeptide substrate R28 of the present invention directly acts on the application in the insect midgut trypsinase vigor that activates bacillus thuringiesis Cry1A class parent toxin in detection.
Wherein, described insect can or come from different population from single species.
The present invention is according to the aminoacid sequence of Cry1A class parent toxin N end, for intestines trypsinase activation site in Cry1A toxoid quilt, at N, hold 28 arginine (seeing accompanying drawing 1), designed a fluorescent polypeptide substrate R28, set up the detection method of insect midgut trypsinase to this fluorogenic substrate, for studying insect midgut, directly acted on the tryptic vigor of Cry1A class parent toxin.3 ' end and the 5 ' two ends at short-movie section polypeptide Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu, add respectively fluorescent quenching group Dabcyl and fluorophor Edans, obtain fluorescent polypeptide substrate R28:Dabcyl-GGERIETGE-Edans, under tryptic effect, the peptide bond that short-movie section polypeptide is located at Arg-Ile (being R-I) is cut off, fluorophor is released, the fluorescent signal that Edans sends is detected by fluorescence microplate reader, tryptic vigor is stronger, the fluorogenic substrate of degraded is more, and the fluorescent signal obtaining is stronger.Present method is by effective utilization of fluorescent polypeptide substrate, improved the susceptibility of detection method, and due to substrate according to insect midgut trypsin acting in the sequences Design at the anatoxic amino acid sites of Cry1A place, make detected result there is clear and definite specific aim, what detect is the tryptic vigor that directly acts on this toxoid albumen, has got rid of the interference of other proteolytic enzyme.
Detection effect, in a method for the insect midgut trypsinase vigor of activation bacillus thuringiesis Cry1A class parent toxin, comprises the following steps:
1) extract enzyme liquid or the midgut epithelial cell brush border membrane vesicle (BBMV, Brush Border Membrane Vesicles) of intestines in lepidopterous insects larva;
2) utilize trypsinase standard substance to set up the typical curve that fluorescent polypeptide substrate R28 enzymolysis of the present invention transforms, clear and definite 1 micromole's fluorogenic substrate transforms the variation of corresponding fluorescent value RFU;
3) measure insect midgut enzyme liquid or the enzymolysis vigor of BBMV to fluorescent polypeptide substrate R28 of the present invention, calculate the tryptic vigor that can directly act on fluorescent polypeptide substrate R28 in midgut juice, thus the enzymolysis ability of clear and definite midgut juice contratoxin.
Wherein, described insect can or come from different population from single species.
Beneficial effect
The present invention is according to the N terminal amino acid sequence of Cry1A class parent toxin, for intestines trypsinase activation site in Cry1A toxoid quilt, be 28 arginine of N end, design a fluorescent polypeptide substrate R28, set up the detection method of insect midgut trypsinase to this fluorogenic substrate.Compare with traditional proteolytic enzyme detection method, it has clear and definite specificity and specific aim, detects the trypsinase vigor that only acts on corresponding fluorogenic substrate R28, and the strong fluorescent signal producing in enzyme digestion reaction has improved the sensitivity detecting greatly.
The anatoxic action target of Cry1A is lepidopterous insects, and the toxin such as Cry1Ac wherein, Cry1Ab are higher to the virulence of insect, are the main toxin compositions of Bt preparation, are also that transgenic anti-insect plants is as the foreign gene mainly proceeding in cotton, corn and paddy rice.The trypsinase fluorogenic substrate R28 for intestines trypsinase activation site in Cry1A toxoid setting up by the present invention and utilize this substrate detect in intestines trypsinase to the anatoxic enzymolysis ability of Cry1A, can, for studying the resistance mechanism of toxin to the insecticidal mechanism of insect and insect contratoxin, there is larger practical value.
Accompanying drawing explanation
The N terminal amino acid sequence figure of Fig. 1 Cry1A parent toxin, and the site of the trypsin digestion in the present invention.
The canonical plotting that Fig. 2 fluorogenic substrate enzymolysis transforms.
Embodiment
Embodiment 1
The first step: the enzyme liquid that extracts intestines in lepidopterous insects larva.
Take lepidopterous insects bollworm as example, get bollworm 5 instar larvaes of the 2nd day, be placed on ice after 30 minutes, with scalpel topping and afterbody, with intestines in tweezers gripping (containing inclusion), in every 3-5 head examination worm, intestines merge as a sample, add the 0.15M sodium chloride solution of 1ml precooling, ice bath homogenate, then homogenate is proceeded to centrifugal 15 minutes of centrifuge tube (12,000g, 4 ℃), get supernatant liquor as enzyme source ,-20 ℃ of storages are standby.
Second step: the BBMV that extracts intestines in lepidopterous insects larva.
Take lepidopterous insects bollworm as example, get bollworm 5 instar larvaes of the 2nd day, be placed on ice after 30 minutes, the head of polypide and afterbody are removed, health is untied along dorsomeson, with intestines in tweezers gripping, remove inclusion simultaneously, in the 0.15M of precooling sodium-chlor, clean, every 10 intestines of merely hitting add 3ml homogenate buffer (300mM N.F,USP MANNITOL, 5mM EGTA and 17mM Tris PH7.5; With front add PMSF a little), ice bath homogenate 30 seconds; Add the about 3.5ml of isopyknic 24mM MgCl2() after mix, ice bath is centrifugal 10 minutes (2500g, 4 ℃) after 30 minutes, supernatant liquor proceeded in new high speed centrifugation pipe to centrifugal 30 minutes of 30,000g; Abandon and be inverted centrifuge tube 10 minutes after supernatant after liquid flows to end, precipitation is resuspended in to HEPES damping fluid (10mM, the HEPES of 800 μ l; 130mM, KCl; 10% glycerine; PH7.4) Hou Yu ?80 ℃ of storages.
The 3rd step: utilize trypsinase standard substance (trypsin, sigma-aldrich company) to set up the typical curve that fluorogenic substrate transforms.
(1) by the fluorogenic substrate R28(Dabcyl-Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu-Edans of 1mg, entrust Qiang Yao bio tech ltd, Shanghai to produce) be dissolved in 690 μ lDMSO, the fluorogenic substrate solution that obtains 1mM, room temperature keeps in Dark Place.
(2) the trypsinase standard substance (trypsin) that Jiang Cong sigma-aldrich company buys are dissolved in 0.1M glycine-sodium hydrate buffer solution of pH10.0, are mixed with the solution of 0.25 milligram every milliliter.The substrate solution of 1mM is diluted to a series of concentration: 0.05mM, 0.25mM, 0.0125mM and 0.0625mM with DMSO.Whole enzyme digestion reaction system is as follows, get 96 hole fluorescence enzyme reaction plates and add therein 200 μ l glycine-sodium hydrate buffer solutions and 5 μ l fluorogenic substrate solution, finally add 10 μ l trypsinase standard solutions, enzyme reaction plate is put into fluorescence microplate reader, design temperature is 30 ℃, reaction times is 100 minutes, and trypsinase standard substance can fully be transformed the fluorogenic substrate of different concns.Adopt end-point method to detect, fluorescence microplate reader sets excitation wavelength lambda ex=336nm in advance, and emission wavelength lambda em=490nm starts first in microplate reader, enzyme reaction plate to be vibrated 1 minute before reading.
(3) using micromole's number of fluorogenic substrate as abscissa, trypsinase standard substance during to the complete enzymolysis of fluorogenic substrate R28 fluorescent value be ordinate, set up the typical curve (seeing accompanying drawing 2) that fluorogenic substrate enzymolysis transforms.According to completely rear corresponding fluorescent value (RFU) the Criterion curve of different concns substrate reactions, obtain transforming the Rfu value that 1 micromole's fluorogenic substrate is corresponding, for calculating the enzyme activity of actual enzyme digestion reaction, 2 results that obtain are for transforming fluorogenic substrate 2.7 * 10 with reference to the accompanying drawings
-5it is 1RFU that micromole can produce fluorescent value.
The 4th step: measure insect midgut enzyme liquid or the enzymolysis vigor of BBMV to fluorogenic substrate, calculate the tryptic vigor that can directly act on fluorogenic substrate R28 in midgut juice or BBMV.
(1) by extracting the insect midgut enzyme liquid or the BBMV that obtain in the first step, react with fluorogenic substrate, whole enzyme digestion reaction system is as follows: get 96 hole fluorescence enzyme reaction plates and add therein 200 μ l glycine-sodium hydrate buffer solutions and 5 μ l fluorogenic substrate solution, finally add 10 μ l midgut juices or BBMV solution, enzyme reaction plate is put into fluorescence microplate reader, adopt KINETIC METHOD to detect.Fluorescence microplate reader sets excitation wavelength lambda ex=336nm in advance, emission wavelength lambda em=490nm, and 30 ℃ of temperature of reaction, 15 minutes reading durations, start first in microplate reader, enzyme reaction plate to be vibrated 1 minute before reading.After reaction, obtain enzymatic reaction slope of a curve, using mRfu/min as unit.The coomassie brilliant blue staining method of employing standard calibrates and participates in the midgut juice of reaction or the protein concentration of BBMV.
(2) according to the relevant regulations of enzyme activity international unit: (be set as 30 ℃ in the present invention) under given conditions, transform the required enzyme amount of 1 micromole's substrate in 1 minute, be called an international unit (IU claims again U).In the present invention, by transforming 1 micromole's fluorogenic substrate R28 in 1 minute, be defined as 1 enzyme activity unit.
Enzymolysis vigor by the midgut juice obtaining in (1) to fluorogenic substrate, the fluorescent value RFU that substitution the 3rd step obtains and the variation relation of fluorogenic substrate R28 mole number, in drawing in midgut juice or BBMV, can directly act on the tryptic vigor of fluorogenic substrate, thereby define midgut juice or the BBMV enzymolysis ability to bacillus thuringiesis Cry1A class parent toxin, and carry out the comparison of different population.
As the SCD strain of bollworm and AY2 strain, extract respectively wherein enzyme liquid and the BBMV of intestines, detect its enzymolysis ability to fluorogenic substrate R28, the enzymolysis vigor result obtaining by this reaction is as table 1, from table, can show that AY2 strain midgut juice is 1.4 times of SCD strain to the enzymolysis vigor of fluorogenic substrate, and the enzymolysis vigor AY2 strain of BBMV only has 0.3 of SCD strain, thereby show the difference of two strains to the enzymolysis ability of Cry1A class parent toxin.
The difference of table 1 different lines enzyme source to fluorogenic substrate enzymolysis ability
Claims (6)
1. for detection of a fluorescent polypeptide substrate R28 for insect midgut trypsinase vigor, it is characterized in that consisting of: fluorescent quenching group-Gly-GLy-Glu-Arg-Ile-Glu-Thr-Gly-Glu-fluorophor.
2. fluorescent polypeptide substrate R28 according to claim 1, is characterized in that described fluorescent quenching group is that Dabcyl and described fluorophor are Edans.
3. fluorescent polypeptide substrate R28 claimed in claim 1 directly acts on the application in the insect midgut trypsinase vigor that activates bacillus thuringiesis Cry1A class parent toxin in detection.
4. application according to claim 3, is characterized in that described insect comes from different population.
5. detect the method for insect midgut enzyme liquid to bacillus thuringiesis Cry1A class parent toxin enzymolysis ability, it is characterized in that comprising the following steps:
1) extract enzyme liquid or the BBMV of intestines in lepidopterous insects larva;
2) utilize trypsinase standard substance to set up its typical curve to fluorescent polypeptide substrate R28 enzyme digestion reaction claimed in claim 1;
3) measure the enzymolysis vigor of insect midgut enzyme liquid to fluorogenic substrate, reference standard curve, calculate the tryptic concentration that can directly act on fluorogenic substrate R28 in midgut juice, thus the enzymolysis ability of clear and definite midgut juice to described bacillus thuringiesis Cry1A class parent toxin.
6. method according to claim 5, is characterized in that described lepidopterous insects is from different population.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624267A (en) * | 2016-02-17 | 2016-06-01 | 中国农业科学院植物保护研究所 | Method for evaluating Bt protein activation actions of midgut digestive enzymes of insects |
| CN111574599A (en) * | 2020-05-18 | 2020-08-25 | 福建农林大学 | Toxin modification method for solving excessive enzymolysis of insecticidal toxin by insect intestinal digestive enzyme |
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Non-Patent Citations (2)
| Title |
|---|
| CRICKMORE ET AL.: "Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins", 《MOCROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS》 * |
| JOHNSTON ET AL.: "Resistance to Bacillus thuringiensis by the Indian meal moth,Plodia interpunctella:comparison of midgut proteinases from susceptible and resistant larvae.", 《JOURNAL OF INVERTEBRATE PATHOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624267A (en) * | 2016-02-17 | 2016-06-01 | 中国农业科学院植物保护研究所 | Method for evaluating Bt protein activation actions of midgut digestive enzymes of insects |
| CN105624267B (en) * | 2016-02-17 | 2019-12-24 | 中国农业科学院植物保护研究所 | Method for evaluating Bt protein activation effect of insect midgut digestive enzyme |
| CN111574599A (en) * | 2020-05-18 | 2020-08-25 | 福建农林大学 | Toxin modification method for solving excessive enzymolysis of insecticidal toxin by insect intestinal digestive enzyme |
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